RESUMO
The pre-mRNA splicing reaction of eukaryotic cells has to be carried out extremely accurately, as failure to recognize the splice sites correctly causes serious disease. The small subunit of the U2AF heterodimer is essential for the determination of 3' splice sites in pre-mRNA splicing, and several single-residue mutations of the U2AF small subunit cause severe disorders such as myelodysplastic syndromes. However, the mechanism of RNA recognition is poorly understood. Here we solved the crystal structure of the U2AF small subunit (U2AF23) from fission yeast, consisting of an RNA recognition motif (RRM) domain flanked by two conserved CCCH-type zinc fingers (ZFs). The two ZFs are positioned side by side on the ß sheet of the RRM domain. Further mutational analysis revealed that the ZFs bind cooperatively to the target RNA sequence, but the RRM domain acts simply as a scaffold to organize the ZFs and does not itself contact the RNA directly. This completely novel and unexpected mode of RNA-binding mechanism by the U2AF small subunit sheds light on splicing errors caused by mutations of this highly conserved protein.
Assuntos
Modelos Moleculares , Proteínas Nucleares/química , Sítios de Splice de RNA , Ribonucleoproteínas/química , Schizosaccharomyces/fisiologia , Dedos de Zinco/fisiologia , Motivos de Aminoácidos , Sítios de Ligação , Análise Mutacional de DNA , Ligação Proteica , Estrutura Quaternária de Proteína , Estrutura Terciária de Proteína , Schizosaccharomyces/química , Fator de Processamento U2AFRESUMO
D-Serine is an essential coagonist with glutamate for stimulation of N-methyl-D-aspartate (NMDA) glutamate receptors. Although astrocytic metabolic processes are known to regulate synaptic glutamate levels, mechanisms that control D-serine levels are not well defined. Here we show that d-serine production in astrocytes is modulated by the interaction between the D-serine synthetic enzyme serine racemase (SRR) and a glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase (GAPDH). In primary cultured astrocytes, glycolysis activity was negatively correlated with D-serine level. We show that SRR interacts directly with GAPDH, and that activation of glycolysis augments this interaction. Biochemical assays using mutant forms of GAPDH with either reduced activity or reduced affinity to SRR revealed that GAPDH suppresses SRR activity by direct binding to GAPDH and through NADH, a product of GAPDH. NADH allosterically inhibits the activity of SRR by promoting the disassociation of ATP from SRR. Thus, astrocytic production of D-serine is modulated by glycolytic activity via interactions between GAPDH and SRR. We found that SRR is expressed in astrocytes in the subiculum of the human hippocampus, where neurons are known to be particularly vulnerable to loss of energy. Collectively, our findings suggest that astrocytic energy metabolism controls D-serine production, thereby influencing glutamatergic neurotransmission in the hippocampus.
Assuntos
Astrócitos/metabolismo , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/metabolismo , Glicólise/fisiologia , Hipocampo/metabolismo , Serina/biossíntese , Trifosfato de Adenosina/genética , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/fisiologia , Animais , Astrócitos/citologia , Gliceraldeído-3-Fosfato Desidrogenase (Fosforiladora)/genética , Hipocampo/citologia , Humanos , Camundongos , Camundongos Endogâmicos ICR , Camundongos Knockout , NADP/genética , NADP/metabolismo , Racemases e Epimerases/genética , Racemases e Epimerases/metabolismo , Serina/genética , Transmissão Sináptica/fisiologiaRESUMO
We developed fluorescent turn-on probes containing a fluorescent nucleoside, 5-(benzofuran-2-yl)deoxyuridine (dU(BF)) or 5-(3-methylbenzofuran-2-yl)deoxyuridine (dU(MBF)), for the detection of single-stranded DNA or RNA by utilizing DNA triplex formation. Fluorescence measurements revealed that the probe containing dU(MBF) achieved superior fluorescence enhancement than that containing dU(BF). NMR and fluorescence analyses indicated that the fluorescence intensity increased upon triplex formation partly as a consequence of a conformational change at the bond between the 3-methylbenzofuran and uracil rings. In addition, it is suggested that the microenvironment around the 3-methylbenzofuran ring contributed to the fluorescence enhancement. Further, we developed a method for detecting RNA by rolling circular amplification in combination with triplex-induced fluorescence enhancement of the oligonucleotide probe containing dU(MBF).
Assuntos
Benzofuranos/química , DNA/química , Desoxiuridina/química , Oligonucleotídeos/química , Uracila/química , Sequência de Bases , DNA de Cadeia Simples/química , Desoxiuridina/análogos & derivados , Corantes Fluorescentes/química , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido Nucleico , RNA/química , Espectrometria de FluorescênciaRESUMO
The family of cytoplasmic polyadenylation element binding proteins CPEB1, CPEB2, CPEB3, and CPEB4 binds to the 3'-untranslated region (3'-UTR) of mRNA, and plays significant roles in mRNA metabolism and translation regulation. They have a common domain organization, involving two consecutive RNA recognition motif (RRM) domains followed by a zinc finger domain in the C-terminal region. We solved the solution structure of the first RRM domain (RRM1) of human CPEB3, which revealed that CPEB3 RRM1 exhibits structural features distinct from those of the canonical RRM domain. Our structural data provide important information about the RNA binding ability of CPEB3 RRM1.
Assuntos
Modelos Moleculares , Fragmentos de Peptídeos/química , Proteínas de Ligação a RNA/química , Regiões 3' não Traduzidas , Sequência de Aminoácidos , Sítios de Ligação , Sequência Conservada , Bases de Dados de Proteínas , Humanos , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Domínios e Motivos de Interação entre Proteínas , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidade Proteica , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , SolubilidadeRESUMO
The intraflagellar transport (IFT) machinery plays a crucial role in the bidirectional trafficking of components necessary for ciliary signaling, such as the Hedgehog, Wnt/PCR, and cAMP/PKA systems. Defects in some components of the IFT machinery cause dysfunction, leading to a wide range of human diseases and developmental disorders termed ciliopathies, such as nephronophthisis. The IFT machinery comprises three sub-complexes: BBsome, IFT-A, and IFT-B. The IFT protein 54 (IFT54) is an important component of the IFT-B sub-complex. In anterograde movement, IFT54 binds to active kinesin-II, walking along the cilia microtubule axoneme and carrying the dynein-2 complex in an inactive state, which works for retrograde movement. Several mutations in IFT54 are known to cause Senior-Loken syndrome, a ciliopathy. IFT54 possesses a divergent Calponin Homology (CH) domain termed as NN-CH domain at its N-terminus. However, several aspects of the function of the NN-CH domain of IFT54 are still obscure. Here, we report the 1H, 15N, and 13C resonance assignments of the NN-CH domain of human IFT54 and its solution structure. The NN-CH domain of human IFT54 adopts essentially the α1-α2-α3-α4-α5 topology as that of mouse IFT54, whose structure was determined by X-ray crystallographic study. The structural information and assignments obtained in this study shed light on the molecular function of the NN-CH domain in IFT54.
Assuntos
Proteínas dos Microfilamentos , Domínios Proteicos , Humanos , Proteínas de Ligação ao Cálcio/química , Proteínas de Ligação ao Cálcio/metabolismo , Calponinas , Proteínas dos Microfilamentos/química , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , SoluçõesRESUMO
Human Transformer2-ß (hTra2-ß) is an important member of the serine/arginine-rich protein family, and contains one RNA recognition motif (RRM). It controls the alternative splicing of several pre-mRNAs, including those of the calcitonin/calcitonin gene-related peptide (CGRP), the survival motor neuron 1 (SMN1) protein and the tau protein. Accordingly, the RRM of hTra2-ß specifically binds to two types of RNA sequences [the CAA and (GAA)(2) sequences]. We determined the solution structure of the hTra2-ß RRM (spanning residues Asn110-Thr201), which not only has a canonical RRM fold, but also an unusual alignment of the aromatic amino acids on the ß-sheet surface. We then solved the complex structure of the hTra2-ß RRM with the (GAA)(2) sequence, and found that the AGAA tetra-nucleotide was specifically recognized through hydrogen-bond formation with several amino acids on the N- and C-terminal extensions, as well as stacking interactions mediated by the unusually aligned aromatic rings on the ß-sheet surface. Further NMR experiments revealed that the hTra2-ß RRM recognizes the CAA sequence when it is integrated in the stem-loop structure. This study indicates that the hTra2-ß RRM recognizes two types of RNA sequences in different RNA binding modes.
Assuntos
Proteínas do Tecido Nervoso/química , Proteínas de Ligação a RNA/química , RNA/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência de Bases , Guanina/química , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Fatores de Processamento de Serina-ArgininaRESUMO
The pre-spliceosomal complex involves interactions between U1 and U2 snRNPs, where a ubiquitin-like domain (ULD) of SF3A1, a component of U2 snRNP, binds to the stem-loop 4 (SL4; the UUCG tetraloop) of U1 snRNA in U1 snRNP. Here, we reported the 1.80 Å crystal structure of human SF3A1 ULD (ULDSF3A1) complexed with SL4. The structural part of ULDSF3A1 (res. 704-785) adopts a typical ß-grasp fold with a topology of ß1-ß2-α1-310a-ß3-ß4-310b-ß5, closely resembling that of ubiquitin, except for the length and structure of the ß1/ß2 loop. A patch on the surface formed by three ULDSF3A1-specific residues, Lys756 (ß3), Phe763 (ß4) and Lys765 (following ß4), contacts the canonical UUCG tetraloop structure. In contrast, the directly following C-terminal tail composed of 786KERGGRKK793 was essentially stretched. The main or side chains of all the residues interacted with the major groove of the stem helix; the RGG residues adopted a peculiar conformation for RNA recognition. These findings were confirmed by mutational studies using bio-layer interferometry. Collectively, a unique combination of the ß-grasp fold and the C-terminal tail constituting ULDSF3A1 is required for the SL4-specific binding. This interaction mode also suggests that putative post-translational modifications, including ubiquitination in ULDSF3A1, directly inhibit SL4 binding.
Assuntos
Ribonucleoproteína Nuclear Pequena U2 , Ubiquitina , Humanos , Ribonucleoproteína Nuclear Pequena U2/genética , Ubiquitina/metabolismo , RNA Nuclear Pequeno/genética , RNA Nuclear Pequeno/metabolismo , RNA , Fatores de Processamento de RNARESUMO
SURP domains are exclusively found in splicing-related proteins in all eukaryotes. SF3A1, a component of the U2 snRNP, has two tandem SURP domains, SURP1, and SURP2. SURP2 is permanently associated with a specific short region of SF3A3 within the SF3A protein complex whereas, SURP1 binds to the splicing factor SF1 for recruitment of U2 snRNP to the early spliceosomal complex, from which SF1 is dissociated during complex conversion. Here, we determined the solution structure of the complex of SURP1 and the human SF1 fragment using nuclear magnetic resonance (NMR) methods. SURP1 adopts the canonical topology of α1-α2-310 -α3, in which α1 and α2 are connected by a single glycine residue in a particular backbone conformation, allowing the two α-helices to be fixed at an acute angle. A hydrophobic patch, which is part of the characteristic surface formed by α1 and α2, specifically contacts a hydrophobic cluster on a 16-residue α-helix of the SF1 fragment. Furthermore, whereas only hydrophobic interactions occurred between SURP2 and the SF3A3 fragment, several salt bridges and hydrogen bonds were found between the residues of SURP1 and the SF1 fragment. This finding was confirmed through mutational studies using bio-layer interferometry. The study also revealed that the dissociation constant between SURP1 and the SF1 fragment peptide was approximately 20 µM, indicating a weak or transient interaction. Collectively, these results indicate that the interplay between U2 snRNP and SF1 involves a transient interaction of SURP1, and this transient interaction appears to be common to most SURP domains, except for SURP2.
Assuntos
Fatores de Processamento de RNA , Ribonucleoproteína Nuclear Pequena U2 , Spliceossomos , Glicina , Humanos , Ligação Proteica , Splicing de RNA , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Spliceossomos/genética , Spliceossomos/metabolismoRESUMO
Matrin-3 is a multifunctional protein that binds to both DNA and RNA. Its DNA-binding activity is linked to the formation of the nuclear matrix and transcriptional regulation, while its RNA-binding activity is linked to mRNA metabolism including splicing, transport, stabilization, and degradation. Correspondingly, Matrin-3 has two zinc finger domains for DNA binding and two consecutive RNA recognition motif (RRM) domains for RNA binding. Matrin-3 has been reported to cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) when its disordered region contains pathogenic mutations. Simultaneously, it has been shown that the RNA-binding activity of Matrin-3 mediated by its RRM domains, affects the formation of insoluble cytoplasmic granules, which are related to the pathogenic mechanism of ALS/FTD. Thus, the effect of the RRM domains on the phase separation of condensed protein/RNA mixtures has to be clarified for a comprehensive understanding of ALS/FTD. Here, we report the 1H, 15N, and 13C resonance assignments of the two RNA binding domains and their solution structures. The resonance assignments and the solution structures obtained in this work will contribute to the elucidation of the molecular basis of Matrin-3 in the pathogenic mechanism of ALS and/or FTD.
Assuntos
Esclerose Lateral Amiotrófica , Demência Frontotemporal , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/metabolismo , Esclerose Lateral Amiotrófica/patologia , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Demência Frontotemporal/patologia , Humanos , Ressonância Magnética Nuclear Biomolecular , RNA/metabolismo , Motivo de Reconhecimento de RNARESUMO
Ribosome biogenesis is a complicated, multistage process coordinated by ribosome assembly factors. Ribosome binding factor A (RbfA) is a bacterial one, which possesses a single structural type-II KH domain. By this domain, RbfA binds to a 16S rRNA precursor in small ribosomal subunits to promote its 5'-end processing. The human RbfA homolog, mtRbfA, binds to 12S rRNAs in the mitoribosomal small subunits and promotes its critical maturation process, the dimethylation of two highly conserved consecutive adenines, which differs from that of RbfA. However, the structural basis of the mtRbfA-mediated maturation process is poorly understood. Herein, we report the 1H, 15N, and 13C resonance assignments of the KH domain of mtRbfA and its solution structure. The mtRbfA domain adopts essentially the same α1-ß1-ß2-α2(kinked)-ß3 topology as the type-II KH domain. Comparison with the RbfA counterpart showed structural differences in specific regions that function as a putative RNA-binding site. Particularly, the α2 helix of mtRbfA forms a single helix with a moderate kink at the Ser-Ala-Ala sequence, whereas the corresponding α2 helix of RbfA is interrupted by a distinct kink at the Ala-x-Gly sequence, characteristic of bacterial RbfA proteins, to adopt an α2-kink-α3 conformation. Additionally, the region linking α1 and ß1 differs considerably in the sequence and structure between RbfA and mtRbfA. These findings suggest some variations of the RNA-binding mode between them and provide a structural basis for mtRbfA function in mitoribosome biogenesis.
Assuntos
Proteínas de Escherichia coli , Proteínas Mitocondriais/química , Ribossomos Mitocondriais , Proteínas de Ligação a RNA/química , Proteínas de Bactérias/química , Proteínas de Escherichia coli/química , Humanos , Ribossomos Mitocondriais/metabolismo , Ressonância Magnética Nuclear Biomolecular , Precursores de RNA/metabolismo , RNA Ribossômico 16S/genética , RNA Ribossômico 16S/metabolismo , Proteínas Ribossômicas/química , Ribossomos/metabolismo , Vitamina B 12/análogos & derivadosRESUMO
The CUG-binding protein 1 (CUG-BP1) is a member of the CUG-BP1 and ETR-like factors (CELF) family or the Bruno-like family and is involved in the control of splicing, translation and mRNA degradation. Several target RNA sequences of CUG-BP1 have been predicted, such as the CUG triplet repeat, the GU-rich sequences and the AU-rich element of nuclear pre-mRNAs and/or cytoplasmic mRNA. CUG-BP1 has three RNA-recognition motifs (RRMs), among which the third RRM (RRM3) can bind to the target RNAs on its own. In this study, we solved the solution structure of the CUG-BP1 RRM3 by hetero-nuclear NMR spectroscopy. The CUG-BP1 RRM3 exhibited a noncanonical RRM fold, with the four-stranded beta-sheet surface tightly associated with the N-terminal extension. Furthermore, we determined the solution structure of the CUG-BP1 RRM3 in the complex with (UG)(3) RNA, and discovered that the UGU trinucleotide is specifically recognized through extensive stacking interactions and hydrogen bonds within the pocket formed by the beta-sheet surface and the N-terminal extension. This study revealed the unique mechanism that enables the CUG-BP1 RRM3 to discriminate the short RNA segment from other sequences, thus providing the molecular basis for the comprehension of the role of the RRM3s in the CELF/Bruno-like family.
Assuntos
Proteínas de Ligação a RNA/química , RNA/química , Motivos de Aminoácidos , Sequência de Bases , Sítios de Ligação , Proteínas CELF1 , Calorimetria , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Sequências Repetitivas de Ácido Nucleico , Homologia de Sequência de AminoácidosRESUMO
In humans, YTH (YT521-B homology) domain containing protein 2 (YTHDC2) plays a crucial role in the phase-shift from mitosis to meiosis. YTH domains bind to methylated adenosine nucleotides such as m6A. In a phylogenic tree, the YTH domain of YTHDC2 (YTH2) and that of the YTH containing protein YTHDC1 (YTH1) belong to the same sub-group. However, the binding affinity of m6A differs between these proteins. Here, we report 1H, 13C and 15N resonance assignment of YTH2 and its solution structure to examine the difference of the structural architecture and the dynamic properties of YTH1 and YTH2. YTH2 adopts a ß1-α1-ß2-α2-ß3-ß4-ß5-α3-ß6-α4 topology, which was also observed in YTH1. However, the ß4-ß5 loops of YTH1 and YTH2 are distinct in length and amino acid composition. Our data revealed that, unlike in YTH1, the structure of m6A-binding pocket of YTH2 formed by the ß4-ß5 loop is stabilized by electrostatic interaction. This assignment and the structural information for YTH2 will provide the insight on the further functional research of YTHDC2.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Adenosina , RNARESUMO
The accurate exclusion of introns by RNA splicing is critical for the production of mature mRNA. U2AF1 binds specifically to the 3´ splice site, which includes an essential AG dinucleotide. Even a single amino acid mutation of U2AF1 can cause serious disease such as certain cancers or myelodysplastic syndromes. Here, we describe the first crystal structures of wild-type and pathogenic mutant U2AF1 complexed with target RNA, revealing the mechanism of 3´ splice site selection, and how aberrant splicing results from clinically important mutations. Unexpected features of this mechanism may assist the future development of new treatments against diseases caused by splicing errors.
Assuntos
Sítios de Splice de RNA/genética , Fator de Processamento U2AF/genética , Fator de Processamento U2AF/metabolismo , Sequência de Bases , Cristalografia por Raios X , Éxons/genética , Humanos , Mutação , Neoplasias/química , Neoplasias/genética , Nucleotídeos , Motivo de Reconhecimento de RNA , Splicing de RNA/genética , Fator de Processamento U2AF/química , Dedos de ZincoRESUMO
The human AU RNA binding protein/enoyl-Coenzyme A hydratase (AUH) is a 3-hydroxy-3-methylglutaconyl-CoA dehydratase in the leucine degradation pathway. It also possesses an RNA-binding activity to AUUU repeats, which involves no known conserved RNA-binding domains and is seemingly unrelated to the enzymatic activity. In this study, we performed mass spectrometric analyses to elucidate the oligomeric states of AUH in the presence and absence of RNA. With a short RNA (AUUU) or without RNA, AUH mainly exists as a trimer in solution. On the other hand, the AUH trimer dimerizes upon binding to one molecule of a long RNA containing 24 repeats of the AUUU motif, (AUUU)(24)A. AUH was crystallized with the long RNA. Although the RNA was disordered in the crystalline lattice, the AUH structure was determined as an asymmetric dimer of trimers with a kink in the alignment of the trimer axes, resulting in the formation of two clefts with significantly different sizes.
Assuntos
Enoil-CoA Hidratase/química , Enoil-CoA Hidratase/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/química , RNA/metabolismo , Cristalização , Humanos , Espectrometria de Massas , Ligação Proteica , Conformação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , UltracentrifugaçãoRESUMO
T cell intracellular antigen-1 (TIA-1), an apoptosis promoting factor, functions as a splicing regulator for the Fas pre-mRNA. TIA-1 possesses three RNA recognition motifs (RRMs) and a glutamine-rich domain. The second RRM (RRM2) is necessary and sufficient for tight, sequence-specific binding to the uridine-rich sequences buried around the 5' splice sites. In the present study, we solved the solution structure of the murine TIA-1 RRM2 by heteronuclear-nuclear magnetic resonance spectroscopy. The TIA-1 RRM2 adopts the RRM fold (betaalphabetabetaalphabeta) and possesses an extra beta-strand between beta2 and beta3, which forms an additional beta-sheet with the C-terminal part of beta2. We refer to this structure as the beta2-beta2' beta-loop. Interestingly, this characteristic beta-loop structure is conserved among a number of RRMs, including the U2AF65 RRM2 and the Sex-lethal RRM1 and RRM2, which also bind to uridine-rich RNAs. Furthermore, we identified a new sequence motif in the beta2-beta2' beta-loop, the DxxT motif. Chemical shift perturbation analyses of both the main and side chains upon binding to the uridine pentamer RNA revealed that most of the beta-sheet surface, including the beta2-beta2' beta-loop, is involved in the RNA binding. An investigation of the chemical shift perturbation revealed similarity in the RNA recognition modes between the TIA-1 and U2AF65 RRMs.
Assuntos
Splicing de RNA , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Sequência Conservada , Cristalografia por Raios X , Humanos , Líquido Intracelular/química , Líquido Intracelular/metabolismo , Líquido Intracelular/fisiologia , Espectroscopia de Ressonância Magnética , Camundongos , Dados de Sequência Molecular , Família Multigênica , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/fisiologia , Conformação Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/fisiologia , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/fisiologia , Soluções , Fator de Processamento U2AF , Propriedades de Superfície , Antígeno-1 Intracelular de Células TRESUMO
The spliceosomal protein p14, a component of the SF3b complex in the U2 small nuclear ribonucleoprotein (snRNP), is essential for the U2 snRNP to recognize the branch site adenosine. The elucidation of the dynamic process of the splicing machinery rearrangement awaited the solution structural information. We identified a suitable complex of human p14 and the SF3b155 fragment for the determination of its solution structure by NMR. In addition to the overall structure of the complex, which was recently reported in a crystallographic study (typical RNA recognition motif fold beta1-alpha1-beta2-beta3-alpha2-beta4 of p14, and alphaA-betaA fold of the SF3b155 fragment), we identified three important features revealed by the NMR solution structure. First, the C-terminal extension and the nuclear localization signal of p14 (alpha3 and alpha4 in the crystal structure, respectively) were dispensable for the complex formation. Second, the proline-rich segment of SF3b155, following betaA, closely approaches p14. Third, interestingly, the beta1-alpha1 loop and the alpha2-beta4 beta-hairpin form a positively charged groove. Extensive mutagenesis analyses revealed the functional relevance of the residues involved in the protein-protein interactions: two aromatic residues of SF3b155 (Phe408 and Tyr412) play crucial roles in the complex formation, and two hydrophobic residues (Val414 and Leu415) in SF3b 155 serve as an anchor for the complex formation, by cooperating with the aromatic residues. These findings clearly led to the conclusion that SFb155 binds to p14 with three contact points, involving Phe408, Tyr412, and Val414/Leu415. Furthermore, to dissect the interactions between p14 and the branch site RNA, we performed chemical-shift-perturbation experiments, not only for the main-chain but also for the side-chain resonances, for several p14-SF3b155 complex constructs upon binding to RNA. These analyses identified a positively charged groove and the C-terminal extension of p14 as RNA-binding sites. Strikingly, an aromatic residue in the beta1-alpha1 loop, Tyr28, and a positively charged residue in the alpha2-beta4 beta-hairpin, Agr85, are critical for the RNA-binding activity of the positively charged groove. The Tyr28Ala and Arg85Ala point mutants and a deletion mutant of the C-terminal extension clearly revealed that their RNA binding activities were independent of each other. Collectively, this study provides details for the protein-recognition mode of p14 and insight into the branch site recognition.
Assuntos
Ressonância Magnética Nuclear Biomolecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteína Nuclear Pequena U2/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo , Spliceossomos/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Sítios de Ligação , Núcleo Celular/química , DNA Complementar/química , Glutationa Transferase/metabolismo , Histidina/metabolismo , Humanos , Hidrólise , Interações Hidrofóbicas e Hidrofílicas , Leucina/química , Modelos Moleculares , Dados de Sequência Molecular , Sinais de Localização Nuclear/química , Fenilalanina/metabolismo , Fosfoproteínas/genética , Ligação Proteica , Conformação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Splicing de RNA , Fatores de Processamento de RNA , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Ribonucleoproteína Nuclear Pequena U2/genética , Ribonucleoproteínas/genética , Homologia de Sequência de Aminoácidos , Spliceossomos/metabolismo , Tripsina/farmacologia , Tirosina/metabolismo , Valina/químicaRESUMO
The SF3a complex, consisting of SF3a60, SF3a66, and SF3a120, in 17S U2 snRNP is crucial to spliceosomal assembly. SF3a120 contains two tandem SURP domains (SURP1 and SURP2), and SURP2 is responsible for binding to SF3a60. We found that the SURP2 fragment forms a stable complex with an SF3a60 fragment (residues 71-107) and solved its structure by NMR spectroscopy. SURP2 exhibits a fold of the alpha1-alpha2-3(10)-alpha3 topology, and the SF3a60 fragment forms an amphipathic alpha helix intimately contacting alpha1 of SURP2. We also solved the SURP1 structure, which has the same fold as SURP2. The protein-binding interface of SURP2 is quite similar to the corresponding surface of SURP1, except for two amino acid residues. One of them, Leu169, is characteristic of SF3a120 SURP2 among SURP domains. Mutagenesis showed that this single Leu residue is the critical determinant for complex formation, which reveals the protein recognition mechanism in the subunit assembly.
Assuntos
Ribonucleoproteína Nuclear Pequena U2/química , Ribonucleoproteínas Nucleares Pequenas/química , Sequência de Aminoácidos , Aminoácidos/química , Análise Mutacional de DNA , Escherichia coli/metabolismo , Humanos , Leucina/química , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Filogenia , Conformação Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de AminoácidosAssuntos
Proteínas Nucleares/química , Domínios e Motivos de Interação entre Proteínas , Proteínas de Ligação a RNA/química , Sequência de Aminoácidos , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Ressonância Magnética Nuclear Biomolecular , Estrutura Secundária de Proteína , Splicing de RNA , Fatores de Processamento de RNA , Alinhamento de SequênciaRESUMO
The spliceosomal protein SF3b49, a component of the splicing factor 3b (SF3b) protein complex in the U2 small nuclear ribonucleoprotein, contains two RNA recognition motif (RRM) domains. In yeast, the first RRM domain (RRM1) of Hsh49 protein (yeast orthologue of human SF3b49) reportedly interacts with another component, Cus1 protein (orthologue of human SF3b145). Here, we solved the solution structure of the RRM1 of human SF3b49 and examined its mode of interaction with a fragment of human SF3b145 using NMR methods. Chemical shift mapping showed that the SF3b145 fragment spanning residues 598-631 interacts with SF3b49 RRM1, which adopts a canonical RRM fold with a topology of ß1-α1-ß2-ß3-α2-ß4. Furthermore, a docking model based on NOESY measurements suggests that residues 607-616 of the SF3b145 fragment adopt a helical structure that binds to RRM1 predominantly via α1, consequently exhibiting a helix-helix interaction in almost antiparallel. This mode of interaction was confirmed by a mutational analysis using GST pull-down assays. Comparison with structures of all RRM domains when complexed with a peptide found that this helix-helix interaction is unique to SF3b49 RRM1. Additionally, all amino acid residues involved in the interaction are well conserved among eukaryotes, suggesting evolutionary conservation of this interaction mode between SF3b49 RRM1 and SF3b145.
Assuntos
Simulação de Acoplamento Molecular , Dobramento de Proteína , Fatores de Processamento de RNA/química , Motivos de Aminoácidos , Humanos , Ressonância Magnética Nuclear Biomolecular , Ligação Proteica , Domínios Proteicos , Fatores de Processamento de RNA/genética , Fatores de Processamento de RNA/metabolismoRESUMO
Tissue-specific alternative pre-mRNA splicing is often cooperatively regulated by multiple splicing factors, but the structural basis of cooperative RNA recognition is poorly understood. In Caenorhabditis elegans, ligand binding specificity of fibroblast growth factor receptors (FGFRs) is determined by mutually exclusive alternative splicing of the sole FGFR gene, egl-15. Here we determined the solution structure of a ternary complex of the RNA-recognition motif (RRM) domains from the RBFOX protein ASD-1, SUP-12 and their target RNA from egl-15. The two RRM domains cooperatively interact with the RNA by sandwiching a G base to form the stable complex. Multichromatic fluorescence splicing reporters confirmed the requirement of the G and the juxtaposition of the respective cis elements for effective splicing regulation in vivo. Moreover, we identified a new target for the heterologous complex through an element search, confirming the functional significance of the intermolecular coordination.